PURPOSE: To quantify the geometrical uncertainties for the heart during radiotherapy treatment of left-sided breast cancer patients and to determine and validate planning organ at risk volume (PRV) margins. METHODS AND MATERIALS: Twenty-two patients treated in supine position in 28 fractions with regularly acquired cone-beam computed tomography (CBCT) scans for offline setup correction were included. Retrospectively, the CBCT scans were reconstructed into 10-phase respiration correlated four-dimensional scans. The heart was registered in each breathing phase to the planning CT scan to establish the respiratory heart motion during the CBCT scan (σ(resp)). The average of the respiratory motion was calculated as the heart displacement error for a fraction. Subsequently, the systematic (Σ), random (σ), and total random (σ(tot)=σ(2)+σ(resp)(2)) errors of the heart position were calculated. Based on the errors a PRV margin for the heart was calculated to ensure that the maximum heart dose (D(max)) is not underestimated in at least 90% of the cases (M(heart) = 1.3Σ-0.5σ(tot)). All analysis were performed in left-right (LR), craniocaudal (CC), and anteroposterior (AP) directions with respect to both online and offline bony anatomy setup corrections. The PRV margin was validated by accumulating the dose to the heart based on the heart registrations and comparing the planned PRV D(max) to the accumulated heart D(max). RESULTS: For online setup correction, the cardiac geometrical uncertainties and PRV margins were ∑ = 2.2/3.2/2.1 mm, σ = 2.1/2.9/1.4 mm, and M(heart) = 1.6/2.3/1.3 mm for LR/CC/AP, respectively. For offline setup correction these were ∑ = 2.4/3.7/2.2 mm, σ = 2.9/4.1/2.7 mm, and M(heart) = 1.6/2.1/1.4 mm. Cardiac motion induced by breathing was σ(resp) = 1.4/2.9/1.4 mm for LR/CC/AP. The PRV D(max) underestimated the accumulated heart D(max) for 9.1% patients using online and 13.6% patients using offline bony anatomy setup correction, which validated that PRV margin size was adequate. CONCLUSION: Considerable cardiac position variability relative to the bony anatomy was observed in breast cancer patients. A PRV margin can be used during treatment planning to take these uncertainties into account.